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Abstract:
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The oceans have proved to be an interminable source of new and
effective drugs. Innumerable studies have proved that specific compounds
isolated from marine organisms have great nutritional and pharmaceutical
value. Polyunsaturated fattyacids (PUFA) in general are known for their
dietary benefits in preventing and curing several critical ailments including
Coronary heart disease (CHD) and cancers of various kinds.
Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) are two
PUFA which are entirely marine in origin – and small Clupeoid fishes like
sardines are known to be excellent sources of these two compounds. In this
study, we selected two widely available Sardine species in the west coast,
Sardinella longiceps and Sardinella fimbriata, for a comparative analysis of
their bioactive properties. Both these sardines are known to be rich in EPA
and DHA, however considerable seasonal variation in its PUFA content was
expected and these variations studied. An extraction procedure to isolate
PUFA at high purity levels was identified and the extracts obtained thus were
studied for anti-bacterial, anti-diabetic and anti-cancerous properties.Samples of both the sardines were collected from landing centre,
measured and their gut content analysed in four different months of the year
– viz. June, September, December and March. The fish samples were
analyzed for fattyacid using FAME method using gas chromatography to
identify the full range of fattyacids and their respective concentration in each
of the samples. The fattyacids were expressed in mg/g meat and later
converted to percentage values against total fatty acids and total PUFA
content. Fattyacids during winter season (Dec-Mar) were found to be
generally higher than spawning season (June-Sept). PUFA dominated the profiles of both species and average PUFA content was also higher during
winter. However, it was found that S. longiceps had proportionately higher
EPA as compared to S. fimbriata which was DHA rich. Percentage of EPA
and DHA also varied across months for both species – the spawning season
seemed to show higher EPA content in S. longiceps and higher DHA content
in S. fimbriata. Gut content analysis indicate that adult S. fimbriata is partial
to zooplanktons which are DHA rich while adult S. longiceps feed mainly on
EPA rich phytoplankton. Juveniles of both species, found mainly in winter,
had a gut content showing more mixed diet. This difference in the feeding
pattern reflect clearly in their PUFA profile – adult S. longiceps, which
dominate the catch during the spawn season, feeding mostly on
phytoplankton is concentrated with EPA while the juveniles which are found
mostly in the winter season has slightly less EPA proportion as compared to
adults. The same is true for S. fimbriata adults that are caught mostly in the
spawning season; being rich in DHA as they feed mainly on zooplankton
while the juveniles caught during winter season has a relatively lower
concentration of DHA in their total PUFA.Various extraction procedures are known to obtain PUFA from fish
oil. However, most of them do not give high purity and do not use materials
indicated as safe. PUFA extracts have to be edible and should not have
harmful substances for applying on mice and human subjects. Some PUFA
extraction procedures, though pure and non-toxic, might induce cis-trans
conversions during the extraction process. This conversion destroys the
benefits of PUFA and at times is harmful to human body. A method free from
these limitations has been standardized for this study. Gas Chromatography
was performed on the extracts thus made to ensure that it is substantially pure. EPA: DHA ratios for both samples were derived - for S. longiceps this
ratio was 3:2, while it was 3:8 for S. fimbriata.Eight common strains of gram positive and gram negative bacterial
strains were subjected to the PUFA extracts from both species dissolved in
acetone solution using Agar Well Diffusion method. The activity was studied
against an acetone control. At the end of incubation period, zones of
inhibition were measured to estimate the activity. Minimum inhibitory
concentration for each of the active combinations was calculated by keeping
p < 0.01 as significant. Four of the bacteria including multi-resistant
Staphylococcus aureus were shown to be inhibited by the fish extracts. It was
also found that the extracts from S. fimbriata were better than the one from S.
longiceps in annihilating harmful bacteria.Four groups of mice subjects were studied to evaluate the antidiabetic
properties of the PUFA extracts. Three groups were induced
diabetes by administration of alloxan tetra hydrate. One group without
diabetes was kept as control and another with diabetes was kept as diabetic
control. For two diabetic groups, a prescribed amount of fish extracts were
fed from each of the extracts. The biochemical parameters like serum
glucose, total cholesterol, LDL & HDL cholesterol, triglycerides, urea and
creatinine were sampled from all four groups at regular intervals of 7 days
for a period of 28 days. It was found that groups fed with fish extracts had
marked improvement in the levels of total LDL & HDL cholesterol,
triglycerides and creatinine. Groups fed with extracts from S. fimbriata seem
to have fared better as compared to S. longiceps. However, both groups did
not show any marked improvement in blood glucose levels or levels of urea.Cell lines of MCF-7 (Breast Cancer) and DU-145 (Prostate Cancer)
were used to analyse the cytotoxicity of the PUFA extracts. Both cell lines
were subjected to MTT Assay and later the plates were read using an ELISA
reader at a wavelength of 570nm. It was found that both extracts had
significant cytotoxic effects against both cell lines and a peak cytotoxicity of
85-90% was apparent. IC50 values were calculated from the graphs and it
was found that S. longiceps extracts had a slightly lower IC50 value indicating
that it is toxic even at a lower concentration as compared to extracts from
S. fimbriata.This study summarizes the bioactivity profile of PUFA extracts and
provides recommendation for dietary intake; fish based nutritional industry
and indigenous pharmaceutical industry. Possible future directions of this
study are also elaborated. |